DE10329195A1 - Slide for fluorescence-based analytical apparatus employing bio-specific sensor chips, comprises a dielectric substrate with a layer containing metallic particles on the surface - Google Patents

Abstract

A slide for fluorescence-based analytical apparatus comprises a dielectric substrate (1) that has a layer on its surface which contains a number of metallic particles (2), where fluorescence is reinforced by marking the sample with fluorescence molecules. A slide for fluorescence-based analytical apparatus comprises a dielectric substrate (1) that has a layer on its surface which contains a number of metallic particles (2), where fluorescence is reinforced by marking the sample with fluorescence molecules. No dimension of any particle exceeds 200 nm. The size is 20-80 nm in each spatial direction. They are ellipsoidal, the aspect ratio being 1:2 to 1:5. The substrate is glass, silicon or a polymer. The layer over the particles has a thickness of 1-10 nm, preferably 2 nm. The metal particles contact the substrate surface. They are fully encapsulated by the layer. The substrate surface is planar. It has parallel projections (4) including an interspace (5). The metal particles are held in the layer only over the projections. Metal particles are held in the layer, in the interspace (5). The layer is applied only in the interspace. It protects the particles from chemical effects. The protective layer (3) comprises a silica compound, a silicon nitride compound or a polymer. It is a polymer matrix (6) permeable to biological molecules. The layer is chemically-functionalized at its surface.

Description

The The invention relates to a slide for fluorescence-based analyzers and relates the problem of low light output, d. H. that usually very small ratio the radiated to the captured photons.

at the investigation of the fluorescence of objects, for example of with fluorescence molecules - so-called Fluorophores, absorbing light of specific wavelengths and as Fluorescent emitting molecules - labeled Samples in fluorescence microscopy or spectroscopy, or at Optical reading of fluorescence-based, biospecific sensor chips Basically there is the problem that only very little of the incident excitation light. absorbed by the fluorophores and converted to fluorescence.

in the In the prior art, this problem is solved by using detection optics used with high numerical apertures. In addition to high production costs you have to The disadvantage of a small field of view, a shallow depth of field and to accept a small working distance. With optoelectronic Alternatively, you can increase the integration time to detect to register the necessary number of fluorescence photons. This elevated but the time required for one Measurement. Furthermore there is the additional problem that that - neither spectrally still spatially completely suppressible - excitation light to a disturbing Background signal leads. Just as disturbing the intrinsic fluorescence of substrates, i. H. Slides, and Optics noticeable. To excite the fluorescence become the objects with light from mercury high pressure lamps illuminated, the power usually does not exceed 100 watts. Theoretically conceivable would be an increase of the lamp power by approx. 5 to 6 orders of magnitude, until an excitation saturation the fluorophore is reached; However, this approach is due the high expected heat generation not practicable.

Out The prior art also discloses that the irradiation of metal particles, with visible light to amplify the electromagnetic Field around the metal particle leads around when the dimensions of the Metal particles small in proportion to the wavelength of the light used for irradiation. This is in the detail for example, in A. Wokaun, "Surface enhancement of optical fields - Mechanisms and applications ", Molecular Physics, 1985, Vol. 56 (1), pages 1-33.

reason for the Field enhancement, which is the bigger the stronger the curvature the surface of the Metal particles are, are collective vibrations of the conduction band electrons of these metal particles, so-called surface plasmons. If you bring now fluorophores in the vicinity - about in a distance that is twice the molecular size - these metal particles, they become as a result of field enhancement by the plasmon field too strong Absorption and subsequent emission of fluorescence photons excited.

This effect is z. B. in the Scriptures US 5,449,918 exploited. There, a fluorescence amplification utilizing chemical sensor is described. This sensor comprises a substrate, on the surface of which a layer of metallic islands is applied. The whole is covered by a chemically selective layer that contains fluorophores and is thinner than 20 nm. This construction is intentionally susceptible to external chemical influences, also the protection against temperature-induced or mechanical stresses, such as rubbing or rinsing, is very low.

In a press release of the Max Planck Society (PRI B12 / C7 / T6 / 2003 (21), ISSN 0170-4656) of March 27 2003 will be a possible Combination of excitation of surface plasmons with fluorescence microscopy in terms of applications for biospecific sensor chips described: A glass carrier is coated with a 50 nm thick layer of gold, this is a about 100 nm thick interaction layer of thiol and a protein applied. The latter serves as a matrix to the DNA strands with a bait function for with dye can dock labeled target sequences. The structure of this arrangement is very complicated and their application is limited. She is too hardly before external chemical or mechanical influences protected. In addition, surface plasmons are formed in the extensive areal gold layer only generated when the layer with light under one for total reflection leading Angle is illuminated.

Of the The invention is therefore based on the object, a fluorescence-enhancing slides for fluorescence-based analyzers to develop in which the fluorescence is enhanced compared to conventional slides.

This object is achieved by a slide comprising a dielectric substrate having a surface and a dielectric layer applied to the surface, wherein the layer contains a plurality of metal particles. In this way the fluorescence of samples labeled with fluorophores is enhanced. The fluorophores are not included in the layer. The direction of incidence of the light is not critical for the generation of surface plasmons, which are the cause of the fluorescence enhancement.

A good fluorescence enhancement you get, if the dimensions of each metal particle in each spatial direction Do not exceed 200 nm, best results will be for Extents between 20 and 80 nm achieved. The absorption spectrum the metal particles overlap in these dimensions with the specific absorption and emission lines most fluorophores. The metal particles can take all possible forms, eg. B. spherical or cuboid However, they are preferably in the form of ellipsoids with an axis ratio between 1: 2 and 1: 5. At the long ends of the ellipsoids is the curvature particularly high, the fluorescence enhancement is therefore particularly close in the vicinity strong.

When Material for the metal particles are preferably gold, silver, copper or aluminum intended. In question is also an alloy that at least one contains these metals to 50%. The metal particles can For example, vapor-deposited on the substrate or by sputtering be applied. It can also self-organizing mechanisms that lead to island formation, be exploited.

The Substrate itself must be dielectric Own properties. Preferably, as the material for the substrate Glass, silicon or a polymer provided.

The Layer has above the metal particles expediently a thickness between 1 nm and 10 nm, preferably 2 nm, which is about twice that Molecular diameter a fluorophore. Are the fluorophores too close to the surface of the Metal particles approach, so decreases the fluorescence-enhancing effect, the same applies too big Distance from the surface the metal particles.

The Metal particles can on the one hand completely be enclosed by the layer or partially with the surface Depending on the type of layer as well as the type of application. For example, if you bring the metal particles first on the surface and covers this afterwards with the layer, so the metal particles are always with the surface of the Substrates are in contact.

In an expedient embodiment The invention is the surface the substrate plan, this allows a simpler preparation of the slide.

In another expedient embodiment the surface of the substrate parallel to each other, web-like elevations on and between them Interspaces. These structures can be, for example by etching, Shape, Injection molding or injection molding be generated, the distance between the surveys is arbitrary and no restrictions subjected.

In A preferred embodiment of the invention are only or predominantly on the strutlike Elevations contain metal particles in the layer. Can be reached for example, by oblique Steaming, here are mainly the bar-shaped Elevations provided with metal particles. These surveys also give Size and shape of the Metal particles in front. This allows a uniform size of metal particles getting produced. When using surveys of various Width can also several discrete metal particle sizes are produced. Of the The essential advantage of this embodiment is that the sizes of the Metal particles exactly on the absorption maxima of certain fluorophores can be matched.

In another embodiment of the invention are only in the interstices of metal particles in the layer included.

In According to a further embodiment of the invention, the layer is only in the gaps applied. This embodiment is particularly for use for the production of fluorescence-based, biospecific sensor chips advantageous because defined in this way from each other Areas on the slide can be produced. It may be appropriate instead of an arrangement with mutually parallel ridge-shaped elevations For example, a grid or honeycomb structure for the surveys provided.

In In a preferred embodiment of the invention, the layer is as for one Variety of biological molecules permeable Polymer matrix configured, for example as a dextran matrix. These Design is particularly with regard to the use for the production fluorescence-based, biospecific sensor chips advantageous because many metal particles and biological molecules embedded in the dextran matrix can be. The fluorescence signal is thereby significantly increased.

In another preferred embodiment of the invention, the layer is designed as a protective layer, so that the metal particles are protected from external chemical and mechanical influences, such as rinsing or rubbing. This is only possible because the fluorophores are not embedded in the layer. Preferred dielectric, ie, insulating materials are silicon oxide compounds such as SiO ₂ or silicon nitride compounds such as Si ₃ N ₄ . These are chemically and mechanically very resistant. Even polymers, ie plastics, come as a protective layer in question. If a protective layer of a silicon compound is used, it can be applied, for example, by evaporation or sputtering. The provided with such a protective layer slide is open to a wide range of applications, he be sitting high stability to external chemical and mechanical influences.

to Improvement of the binding of biomolecules is in a further embodiment the invention chemically functionalizes the layer on its surface, for example, with epoxy, amino or aldehyde groups.

The Invention will be explained in more detail below in some embodiments. In the corresponding Drawings show

1 the simplest embodiment of a slide according to the invention with a protective layer of silicon dioxide;

2 to 4 Modifications thereof,

5 a slide in which metal particles are embedded in a polymer matrix, and

6 the section of a slide from the 1 - 5 in plan view when used to make biospecific fluorescence-based sensor chips.

1 shows a slide according to the invention in the simplest embodiment. As a substrate 1 Glass is used on the substrate 1 are metal particles 2 applied by one as a protective layer 3 configured layer are covered. The diameter of the metal particles 2 in the plane of the substrate lies between 20 and 80 nm. The protective layer 3 above the metal particles 2 is about 2 nm thick. It consists of silicon dioxide, but other silicon compounds are conceivable.

In 2 indicates the surface of the substrate 1 bar-shaped, parallel elevations 4 on. Between the surveys 4 there are gaps 5 , The metal particles 2 , preferably of precious metals such as gold or silver, are on the surface of the substrate 1 both on the surveys 4 as well as in the interstices 5 , All metal particles 2 are covered by a continuous protective layer 3 covered. It is not mandatory that the web-shaped elevations 4 are arranged parallel to each other. Depending on the application, another arrangement, for example, a grid-shaped, may be advantageous. The surveys 4 For example, can be prepared by substrate material from the spaces 5 is etched away.

In 3 a slide is shown in which the ridge-shaped elevations 4 stand closer together and only on these metal particles 2 are applied, which can be achieved for example by oblique vapor deposition. The upper surfaces of the webs so give the shape and size of the metal particles 2 in front. In this way, the size of the metal particles 2 by specifying the land size exactly on the absorption maxima of specific fluorophores to be used, are tuned.

In 4 are the metal particles 2 in the protective layer 3 and are completely enclosed by it. Moreover, only in the interstices 5 metal 2 contain, in this way are the metal particles 2 through the web-shaped elevations 4 well protected against mechanical influences such as rubbing or rinsing.

In the in 5 As shown in the embodiment of the invention, the layer is a polymer in the form of a polymer matrix 6 used. In question comes here, for example, dextran. The metal particles 2 are in this polymer matrix permeable to fluorescently labeled biomolecules 6 embedded image, wherein the average distance between the - statistically distributed in the polymer matrix - metal particles and applied in the course of application to the slide fluorescently labeled biomolecules is also about 2 nm. As material for the metal particles 2 Gold is preferred because of its chemical resistance. The metal particles are so very accessible to the fluorophores.

In 6 It is shown how a fluorescence-enhancing slide can be used for the production of fluorescence-based biospecific sensor chips. Shown is a section of the top view of a slide. The areas D1-D6 are approximately 20 μm × 20 μm in size - the distances between the elevations 4 are usually much smaller, but can also be chosen according to the dimensions of the areas - and are each associated with a variety of molecules with bait function, such as selected DNA sequences.

1

substratum

2

metal

3

protective layer

4

surveys

5

interspaces

6

polymer matrix

D1, ..., D6

areas

Claims (21)

Microscope slides for fluorescence-based analyzers, comprising - a dielectric substrate ( 1 ) having a surface and - a surface-applied dielectric layer in which a plurality of metal particles ( 2 ), thereby enhancing the fluorescence of samples labeled with fluorescent molecules. Slide according to claim 1, characterized in that the largest dimension of each metal particle ( 2 ) in each spatial direction is not more than 200 nm. Slide according to one of the preceding claims, characterized in that the extent of each metal particle ( 2 ) is in each spatial direction between 20 nm and 80 nm. Slide according to one of the preceding claims, characterized in that the metal particles ( 2 ) have the shape of ellipsoids. slides according to claim 4, characterized in that the ratio of the axes of each Ellipsoids is between 1: 2 and 1: 5. Slide according to one of the preceding claims, characterized in that as the material for the metal particles ( 2 ) Gold (Au), silver (Ag), copper (Cu), aluminum (Al) or an alloy containing at least one of the aforementioned metals at 50% is provided. Slide according to one of the preceding claims, characterized in that the material used for the substrate ( 1 ) Glass, silicon or a polymer is provided. Slide according to one of the preceding claims, characterized in that the layer above the metal particles ( 2 ) has a thickness between 1 nm and 10 nm, preferably 2 nm. Slide according to one of the preceding claims, characterized in that the metal particles are bonded to the surface of the substrate ( 1 ) stay in contact. Slide according to one of Claims 1 to 9, characterized in that the layer contains the metal particles ( 2 ) completely encloses. Slide according to one of the preceding claims, characterized in that the surface of the substrate ( 1 ) is plan. Slide according to one of claims 1 to 11, characterized in that the surface of the substrate ( 1 ) parallel, ridge-shaped elevations ( 4 ) and between these spaces ( 5 ) having. Slide according to claim 13, characterized in that only above the elevations ( 4 ) Metal particles ( 2 ) are contained in the layer. Slide according to claim 13, characterized in that only in the spaces ( 5 ) Metal particles ( 2 ) are contained in the layer. Slide according to claim 15, characterized in that the layer is only in the interstices ( 5 ) is applied. Slide according to one of the preceding claims, characterized in that the layer is used as a protective layer ( 3 ), wherein the metal particles ( 2 ) through the protective layer ( 3 ) are protected from external chemical influences. Slide according to one of the preceding claims, characterized in that as material for the protective layer ( 3 ) a silicon oxide compound, a silicon nitride compound or a polymer is provided. Slide according to one of Claims 1 to 15, characterized in that the layer is a polymer matrix permeable to a large number of biological molecules ( 6 ) is configured. Slide according to claim 18, characterized in that as material for the polymer matrix ( 6 ) Dextran is used. slides according to one of the preceding claims, characterized that the layer on their surface is chemically functionalized. Use of a slide according to one of the preceding claims Production of fluorescence-based, biospecific sensor chips.